JPH0636746B2 - Method for producing L-glutamic acid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] L-glutamic acid is widely used as a food material such as seasonings and as a raw material for various chemical products. The present invention is L
-It relates to a method for efficiently producing glutamic acid.

[Conventional technology]

L-glutamic acid is generally produced by a fermentation method.
Various studies have been carried out to improve the productivity of L-glutamic acid. For example, in order to increase the amount of L-glutamic acid accumulated in the fermentation liquor, sugar as a raw material is additionally added, or L-glutamic acid-producing bacteria ( Hereinafter, it is sometimes referred to as a fermenting bacterium.) Techniques such as a method for sufficiently supplying oxygen required by the fermenting bacterium have been developed. Also, in order to save the raw material and time required for seed culture of fermenting bacteria, a method has been developed in which the bacterial cells that have once undergone fermentation production are recovered from the fermentation liquid and suspended in the next medium for reuse. .

[Problems to be solved by the invention]

In any case, the conventional methods have tried to improve productivity by increasing the concentration of L-glutamic acid accumulated in the fermentation broth. However, such a method has a problem that the productivity of L-glutamic acid of the fermenting bacterium in the latter half of the fermentation is reduced, so that the productivity cannot be greatly improved.

In addition, when the microbial cells that have once been fermented are reused, the L-glutamic acid-producing ability of this bacterium is reduced, and it is not possible to efficiently produce L-glutamic acid. This method was also problematic in that it was easily contaminated by miscellaneous cells during the period from the recovery of the bacterial cells to the suspension in the next medium.

[Means for solving problems]

The present inventors have conducted various studies to solve such problems and develop a method for efficiently producing L-glutamic acid, and as a result, in the conventional method for recovering and reusing the bacterial cells, the latter half of fermentation was performed. It has been found that the productivity of L-glutamic acid is lowered because the cells are exposed to high concentration of L-glutamic acid and high osmotic pressure. Then, by avoiding exposing the cells to a high concentration of glutamic acid or high osmotic pressure by extracting a part of the fermentation broth during the fermentation and replenishing a new medium, the cells can produce a high L-glutamic acid production capacity for a long period of time. Based on this finding, the present invention was completed based on the finding that L-glutamic acid can be efficiently produced.

That is, the present invention relates to at least a reaction tank, a bacterial cell separation device connected to the reaction tank to centrifuge bacterial cells from a reaction liquid discharged therefrom, and a supernatant discharged from the bacterial cell separation device connected thereto. Using a device having a glutamic acid separation device for separating L-glutamic acid from a liquid and a pipe for returning the bacterial cell liquid discharged from the bacterial cell separation device to the reaction tank, the reaction tank is reacted with L-glutamic acid-producing bacteria and biotin L-glutamic acid production reaction with 50-500γ / in fatty acid and fatty acid or its ester as 0.05-0.5% in reaction liquid or penicillin as 1-10 U / m in reaction liquid and substrate solution Then, a part of the reaction solution in the reaction tank is extracted and the bacterial cells are centrifuged by the bacterial cell separation device, and the separated bacterial cell liquid is returned to the reaction tank, while the bacterial cells are separated. did The supernatant is sent to the glutamic acid separation device, where L-glutamic acid is separated,
In the reaction vessel, biotin is 50-500γ /, fatty acid or its ester is 0.05-0.5%, or penicillin is 1-1.
The substrate solution containing 0 U / m is supplied to replenish the reaction to continue the reaction, and the amount of liquid exchanged at that time is 5% or more of the total amount of reaction liquid per hour, and the above operations are performed continuously or intermittently. The present invention relates to a method for producing L-glutamic acid by a fermentation method, which comprises repeatedly adjusting the L-glutamic acid concentration of a reaction solution in a reaction tank to 8 to 15%.

The L-glutamic acid-producing bacterium used in the method of the present invention is not particularly limited, and an ordinary fermenting bacterium used for L-glutamic acid fermentation may be used. Examples include Brevibacterium lactofermentum ATCC13
869, Brevibacterium flavum ATCC14067, Corynebacterium glutamicum ATCC13032, etc. can be mentioned.

The substrate solution is a solution of a known substrate (carbon source) for fermentation of L-glutamic acid, such as glucose, sucrose, molasses, starch hydrolyzate, ethanol, organic acid, and hydrocarbon.

This substrate solution contains ordinary medium components for L-glutamic acid fermentation, such as ammonium sulfate, ammonium nitrate, ammonium chloride, nitrogen sources such as ammonia, urea, amino acids, 1 potassium phosphate, 2 potassium phosphate, 1 sodium phosphate, and sulfuric acid. Inorganic salts such as magnesium, iron salts and manganese salts, organic nutrients such as soybean protein hydrolysates and the like can be appropriately added. Concentrations of nitrogen source, inorganic salts, organic nutrients and the like may be similar to those in a normal medium.

In the method of the present invention, in addition to the above substrate, biotin or its derivative and fatty acid or its ester or penicillin or its derivative are added.

The fatty acid is a saturated higher fatty acid having 12 to 18 carbon atoms, and its ester is glycerol ester, sorbitan ester, sucrose ester, polyethylene glycol ether, polyoxyethylene sorbitan ester and the like.

Biotin or a derivative thereof is effective because the fermentation bacterium retains the L-glutamic acid-producing ability for a long period of time.
The amount of addition is such that the concentration in the reaction solution is about 50γ / to 500γ /.

In order to efficiently produce L-glutamic acid, it is effective to add fatty acid or its ester or penicillin or its derivative in addition to biotin. In the case of fatty acid or its ester, the concentration in the reaction solution is preferably about 0.05% to 0.5%, and in the case of penicillin or its derivative, the concentration in the reaction solution is 1 U.
/ M to 10 U / m is suitable.

Each of the above components may be directly charged into the reaction tank separately from the substrate solution. In particular, it is preferable that biotin or its derivative, fatty acid or its derivative, and penicillin or its derivative can be added separately so that the concentration in the reaction solution can be adjusted to an appropriate range.

An outline of an example of an apparatus used in the method of the present invention is shown in FIG.

The reaction tank is for carrying out the L-glutamic acid production reaction, and is provided with a temperature control mechanism, a ventilation mechanism for the reaction solution, and a pH control mechanism, as well as an inlet for the bacterial cells and substrate solution and an outlet for the reaction solution. There is a need. In addition, a stirrer and various instruments such as a dissolved oxygen concentration meter, a substrate concentration meter, a concentration meter for various medium components, an L-glutamic acid concentration meter, and a liquid level gauge may be appropriately provided. The reaction tank may have any shape, for example, a cylindrical shape or a box shape. A conventional fermentation tank can be used for such a reaction tank.

Since the substrate usually remains in the reaction liquid extracted from the reaction tank, it is preferable that the substrate is once put in an intermediate tank and the substrate is modified therein to be converted into L-glutamic acid. The intermediate tank is preferably equipped with a temperature and pH adjusting mechanism and a ventilation mechanism, and in addition, the equipment and the like provided in the reaction tank are appropriately added if necessary. As the intermediate tank, a complete mixing tank or a retention tube type reaction tank can be used. The intermediate tank is highly necessary in the system in which the substrate solution is continuously added to continuously withdraw the reaction solution, and conversely, in the system in which these are intermittently performed, the intermediate tank may be omitted in some cases.

A centrifugal separator is used as the cell separation device. However, it is necessary to take care so that the temperature of the cells does not rise during cell separation. A continuous centrifuge can also be preferably used in the method of the present invention, but there is also a centrifuge of the type in which cells are heated during the separation. In such a case, it is necessary to cool the reaction solution in advance and then separate the cells.

The microbial cell liquid side discharged from the microbial cell separator is piped so as to return to the reaction tank, while the supernatant liquid side is piped to the glutamic acid separator.

The glutamic acid separation device may be an ordinary device, and for example, a combination of a crystallizer and a crystal separator, an ion exchange resin tower, an electrodialysis device and the like may be used.

In addition to these, a substrate solution preparation tank, its storage tank, a seed culture tank, and auxiliary equipment necessary for this are appropriately provided.

When L-glutamic acid is produced using such an apparatus, first, the cells and the substrate solution are put in a reaction tank and the mixture is added to
After reacting for about 30 hours, the bacterial cells grow to the extent necessary to efficiently produce L-glutamic acid. If this time is too short, the amount of bacterial cells will be insufficient and the subsequent efficient L-
Production of glutamic acid becomes difficult, while if it is too long, the substrate will be consumed up and the L-glutamic acid-producing ability of the cells will decrease.

Therefore, a part of the reaction solution is sent from the reaction tank to the intermediate tank, while the substrate solution is sent from the substrate storage tank to the reaction tank to supplement the decrease in the solution amount and continue the reaction. The amount of the liquid to be exchanged is set to 5% or more, preferably 10% or more of the total amount of the reaction liquid in one hour. If the amount of this exchange liquid is too small, the concentration of L-glutamic acid in the reaction tank becomes too high, or the osmotic pressure becomes too high, and the L-glutamic acid-producing ability of the cells decreases. The exchange of liquid may be continuous or intermittent.

The substrate concentration of the reaction solution in the reaction tank is controlled to be about 0.1 to 5%. If it is less than 0.1%, the rate of qualification of the substrate is lowered and the production rate of L-glutamic acid is lowered. On the other hand, if it exceeds 5%, inhibition of substrate concentration or inhibition due to increase in osmotic pressure appears, and the L-glutamic acid production rate also decreases.

The L-glutamic acid concentration of this reaction solution is controlled to be about 8 to 15%. When it is 8% or less, the efficiency of separating L-glutamic acid from the supernatant becomes poor, while 15%
When it exceeds, the L-glutamic acid-producing ability of the fermenting bacterium is inhibited and the production rate of L-glutamic acid decreases.

The osmotic pressure of the reaction solution depends on the concentration of inorganic salt, the concentration of ammonia, L
-It increases as the concentration of glutamic acid, the concentration of substrate, the concentration of nitrogen source, etc. increase. If the osmotic pressure exceeds 2000 osomomol, the production rate of L-glutamic acid decreases, so the osmotic pressure is controlled to be less than this.

The pH of the reaction solution is preferably adjusted to the position where the fermenting bacterium produces L-glutamic acid at the maximum rate, and this pH is usually controlled so as to fall within the range of 7 to 8. Also, the temperature is preferably adjusted to a range in which the fermenting bacterium can maintain a high L-glutamic acid producing ability for a long period of time,
It is usually maintained at 30 to 40 ° C. It is generally preferable to rapidly grow the bacteria at 30 to 35 ° C during the growth phase from the main reaction tank to the start of liquid feeding, and maintain the temperature at 35 to 40 ° C after the liquid feeding is started.

During the reaction, the reaction solution is aerated and, if necessary, stirred to maintain the reaction solution under aerobic conditions. The dissolved enzyme partial pressure of the reaction solution is preferably maintained at about 0.1 to 10%. When it is 0.1% or less, an organic acid such as lactic acid or an amino acid other than L-glutamic acid such as L-alanine is produced to reduce the production rate of L-glutamic acid. On the other hand, if it exceeds 10%, the oxidation of lipids and the like, which are the constituents of bacterial cells, is promoted, and the L-glutamic acid productivity is reduced. The dissolved oxygen partial pressure can be measured with a general dissolved oxygen electrode of galvanic type, polaro type, etc., and is a value when the air is saturated to 21%.

In addition, it is preferable to adjust the concentration of biotin or the like during the reaction so as to be maintained in an appropriate range.

In the intermediate tank, the substrate remaining in the reaction solution is assimilated, and for this purpose, this tank is maintained at about 30 to 40 ° C., and if necessary, aeration and stirring are performed. When the cell separation device is a continuous centrifugal type, it is preferable that the substrate is assimilated and then cooled to about 5 to 10 ° C. and then sent to the cell separation device.

The microbial cell liquid separated by the microbial cell separation device is returned to the reaction tank, but it is possible to continue operation for a longer period by withdrawing part of it and supplementing the microbial cells obtained by separate seed culture. Become.

On the other hand, the supernatant is sent to a glutamic acid separation device where L
-Separate glutamic acid.

[Action]

In the method of the present invention, when a microorganism is mixed with a raw material serving as a substrate and a reaction for producing L-glutamic acid is carried out,
Before the accumulation of L-glutamic acid and other substances that inhibit the L-glutamic acid formation reaction and the deterioration of the environment occur, a part of the reaction solution is replaced, and the microorganisms always have G ^H.
By maintaining the environmental conditions more suitable for production, separating the microorganisms discharged from the reaction tank to replace the reaction solution with the culture solution containing the product so as not to be contaminated by miscellaneous bacteria, and returning it to the reaction tank again. It makes it possible to efficiently produce L-glutamic acid while maintaining the L-glutamic acid producing activity of the microorganism for a long time.

〔Example〕

 The present invention will be described in more detail below with reference to examples.

Example 1. Glucose _{30g /, KH 2 PO 4 1g} /, MgSO 4 · 7aq0.4
g /, Urea 4g /, FeSO ₄ / 7aq 20mg /, MnSO ₄・ 4
A medium 50 containing aq 20 mg /, soybean protein acid hydrolyzate 5 m /, and biotin 300 μg / was placed in an 80-volume small fermenter and sterilized by heating at 120 ° C. for 10 minutes. After cooling
Corynebacterium glutamicum ATCC13032 was inoculated and seed-cultured for 24 hours at 31.5 ° C. Glucose 100 g /, KH ₂ PO ₄ 1 g /, soybean protein acid hydrolyzate 20 m /, biotin 300 μg /
A substrate solution 950 containing 1 g / g of MgSO ₄ .7aq was placed in a previously sterilized 1.5 k reaction vessel and kept at 31.5 ° C. The sterilized air was aerated for 1 k / min, and the pH was kept at 7.5 with NH ₃ gas, and the mixture was stirred at 250 rpm. Five hours after the start, 10 U of penicillin G dissolved in 10 sterile water was added.
It was added so as to have a concentration of m. After continuing the reaction for another 5 hours, the reaction solution was heated at 31.5 ° C at a speed of 100 / hr.
It passed through the retention tube which was kept warm. Then, it was cooled by passing through a heat exchanger until the liquid temperature reached 10 ° C., and led to a Westfalia continuous centrifuge. The liquid was separated into a light liquid containing no cells and a heavy liquid containing cells so that the liquid ratio was 9: 1, the heavy liquid was returned to the reaction tank, and the light liquid was put into a neutralization crystallization tank. Glucose 150 g /, KH ₂ PO ₄ 1 g /, soybean protein acid hydrolyzate 5 m /, biotin 300 μg /
, MgSO ₄ .7ap 1 g /, and a penicillin G 10 U / m substrate solution were continuously supplied at a rate of 90 per hour to keep the liquid volume in the reaction tank constant. After this culture was continued for 48 hours, it was sent to the neutralization crystallization tank, including the culture solution in the reaction tank, to obtain a total of 5300 L-glutamic acid-containing solutions. From this liquid, 515 kg of L-glutamic acid was obtained.

On the other hand, the seed culture solution 50 prepared in the same manner was used as glucose 10
0 g /, KH ₂ PO ₄ 1 g /, soy protein hydrolyzate 20
m /, biotin 300μg /, MgSO ₄ · 7aq1g /
The substrate solution 950 containing the above was placed in a pre-sterilized 1.5 k volume reaction tank and kept at 31.5 ° C. The sterilized air was aerated for 1 k / min, and the pH was maintained at 7.5 with NH ₃ gas, and the mixture was stirred at 250 rpm. Five hours after the initiation, penicillin G dissolved in 10 sterile water was added so as to have a concentration of 10 U / m. After reacting for another 5 hours, glucose 500 was added without removing the reaction solution from the reaction tank.
g /, KH ₂ PO ₄ 1 g /, soybean protein acid hydrolyzate 5 m
/, Biotin _{300μg /, MgSO 4 · 7ap1g /} ,
Substrate solution containing penicillin G10U / m 15k / h
Each was continuously supplied, and the reaction was continued for up to 48 hours. As a result, a reaction solution containing 1550 L-glutamic acid was obtained, and 192 kg of L-glutamic acid was obtained from this reaction solution.

〔The invention's effect〕

By the method of the present invention, L-glutamic acid can be continuously produced at a high production rate. Further, since the amount of waste bacterial cells discharged is small, the labor and cost of the treatment can be saved. By adopting the continuous production system, the labor load can be reduced, the device can be efficiently used, and the cost can be largely reduced as a whole.

[Brief description of drawings]

FIG. 1 is a flow sheet showing an outline of an example of an apparatus used in the method of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of front page (56) References Japanese Patent Publication No. 55-24876 (JP, B2) European Patent Publication 139592 (EP, A)

Claims (2)

[Claims] 1. At least a reaction tank, a bacterial cell separation device which is connected to the reaction tank and centrifuges bacterial cells from a reaction solution discharged therefrom, and a supernatant liquid which is connected to the bacterial cell separation apparatus and discharged therefrom. Using a device having a glutamic acid separation device for separating L-glutamic acid from the reaction product and a pipe for returning the bacterial cell liquid discharged from the bacterial cell separation device to the reaction tank, the reaction solution containing L-glutamic acid-producing bacteria and biotin in the reaction tank. The concentration of the fatty acid or its ester is 0.05 to 0.5% as the concentration in the reaction solution or 0.05 to 0.5% as the concentration in the reaction solution or 1 to 10 U / m as the concentration in the reaction solution and the substrate solution is added to carry out the L-glutamic acid formation reaction. Then, a part of the reaction solution in the reaction tank was extracted and the bacterial cells were centrifuged by the bacterial cell separation device, and the separated bacterial cell liquid was returned to the reaction tank, while the bacterial cells were separated. Supernatant The is the glutamate separation device to send it where it separates the L- glutamic acid, wherein 0.05% to 0.5% of 50～500Ganma / and fatty acid or ester thereof biotin in the reaction vessel or penicillin 1-1
The substrate solution containing 0 U / m is replenished to continue the reaction, and the amount of liquid exchanged at that time is 5% of the total amount of the reaction liquid per hour.
As described above, a method for producing L-glutamic acid by a fermentation method, which comprises repeating the above-mentioned operations continuously or intermittently to adjust the L-glutamic acid concentration of the reaction solution in the reaction tank to 8 to 15% 2. A post-bacterial cell separation device in which an intermediate tank is provided between the reaction tank and the bacterial cell separation device, and the reaction liquid extracted from the reaction tank is further reacted there to assimilate the remaining substrate. The method according to claim 1, wherein the bacterial cells are separated by